Estudo da produção de ligninases e celulases pelo fungo Pycnoporus sanguineus MCA 16 e uso dos extratos enzimáticos na sacarificação do bagaço de cana-de-açúcar para produção de bioetanol
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Data
2018-07-27
Autores
Pereira, Josiani de Cassia
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Universidade Estadual Paulista (Unesp)
Resumo
No presente trabalho, o fungo Pycnoporus sanguineus MCA16 foi cultivado em diferentes substratos lignocelulósicos, por fermentação em estado sólido, para obtenção de celulases, xilanases e ligninases. Estas enzimas foram empregadas na sacarificação do bagaço de cana-de-açúcar submetido a pré-tratamento hidrotérmico alcalino realizado na presença ou ausência de agentes oxidantes auxiliares na remoção da lignina, como KMnO4, H2O2, ZnO e TiO2.com o intuito de auxiliar na remoção da lignina e desestruturação da lignocelulose. Além disso, as ligninases produzidas pelo bioprocesso foram estudadas quanto a degradação de compostos fenólicos totais liberados durante a etapa de sacarificação enzimática a fim de que promova um tratamento enzimático. Dentre todos os substratos lignocelulósicos utilizados, a mistura de farelo de trigo e farelo de soja levou à uma maior produção da maioria das enzimas testadas em 96h à 40°C. Após isso as soluções enzimáticas foram aplicadas na sacarificação enzimática do bagaço de cana-de-açúcar pré-tratado, tanto hidrotérmico alcalino quanto empregando agentes oxidantes, utilizando ferramenta de planejamento estatístico para avaliar a influência de diferentes variáveis. Diante de todos os agentes oxidantes avaliados, H2O2 destacou-se obtendo as maiores liberações de glicose durante a sacarificação enzimática, com 250 unidades de endoglucanase/g de celulose com 11% de bagaço de cana-de-açúcar pré-tratado com 2,5% de H2O2 durante 106 horas à 50°C. A fermentação alcoólica do hidrolisado nas condições de 288,3 unidades de endoglucanase/g de celulose com 13,5% de bagaço de cana-de-açúcar sob pré-tratamento hidrotérmico alcalino durante 130 horas à 57°C gerou uma conversão de glicose à etanol de 73,5%. Após a sacarificação enzimática, a concentração de compostos fenólicos sofreu uma redução de aproximadamente 90% enquanto que lacase e lignina peroxidase apresentaram uma ativação enzimática de 145% e 226%, respectivamente. Além disso, ácidos orgânicos gerados pela degradação da lignina exerceram influência sobre estas enzimas, ativando-as.
In the present work, the fungus Pycnoporus sanguineus MCA16 was grown on different lignocellulosic substrates by solid-state fermentation to obtain cellulases, xylanases and ligninases. These enzymes were used in saccharification of sugarcane bagasse submitted to alkaline hydrothermal pretreatment carried out in the presence or absence of oxidizing agents such as KMnO4, H2O2, ZnO and TiO2 in order to assist in the lignin removal and lignocellulose disruption. In addition, the ligninases produced by the bioprocess were studied for the degradation of total phenolic compounds released during the enzymatic saccharification step in order to promote an enzymatic treatment. Among all the lignocellulosic substrates used, the mixture of wheat bran and soybean meal led to a higher production of most of the enzymes tested in 96h at 40 °C. After that, the enzymatic solutions were applied in the enzymatic saccharification of pretreated sugarcane bagasse, both alkaline hydrothermal and using oxidizing agents, using a statistical planning tool to evaluate the influence of different variables. In view of all the oxidizing agents evaluated, H2O2 was selected by obtaining the highest glucose releases during the enzymatic saccharification with 250 units of endoglucanase/g cellulose with 11% sugarcane bagasse pre-treated with 2.5 % H2O2 for 106 hours at 50 °C. The alcoholic fermentation of the hydrolyzate under the conditions of 288.3 units of endoglucanase/g cellulose with 13.5% sugarcane bagasse under hydrothermal alkaline pre-treatment for 130 hours at 57 °C generated a conversion of glucose to 73.5% ethanol. After enzymatic saccharification, the concentration of phenolic compounds decreased by approximately 90% while laccase and lignin peroxidase showed an enzymatic activation of 145% and 226%, respectively. In addition, organic acids generated by the degradation of lignin exerted influence on these enzymes, activated them.
In the present work, the fungus Pycnoporus sanguineus MCA16 was grown on different lignocellulosic substrates by solid-state fermentation to obtain cellulases, xylanases and ligninases. These enzymes were used in saccharification of sugarcane bagasse submitted to alkaline hydrothermal pretreatment carried out in the presence or absence of oxidizing agents such as KMnO4, H2O2, ZnO and TiO2 in order to assist in the lignin removal and lignocellulose disruption. In addition, the ligninases produced by the bioprocess were studied for the degradation of total phenolic compounds released during the enzymatic saccharification step in order to promote an enzymatic treatment. Among all the lignocellulosic substrates used, the mixture of wheat bran and soybean meal led to a higher production of most of the enzymes tested in 96h at 40 °C. After that, the enzymatic solutions were applied in the enzymatic saccharification of pretreated sugarcane bagasse, both alkaline hydrothermal and using oxidizing agents, using a statistical planning tool to evaluate the influence of different variables. In view of all the oxidizing agents evaluated, H2O2 was selected by obtaining the highest glucose releases during the enzymatic saccharification with 250 units of endoglucanase/g cellulose with 11% sugarcane bagasse pre-treated with 2.5 % H2O2 for 106 hours at 50 °C. The alcoholic fermentation of the hydrolyzate under the conditions of 288.3 units of endoglucanase/g cellulose with 13.5% sugarcane bagasse under hydrothermal alkaline pre-treatment for 130 hours at 57 °C generated a conversion of glucose to 73.5% ethanol. After enzymatic saccharification, the concentration of phenolic compounds decreased by approximately 90% while laccase and lignin peroxidase showed an enzymatic activation of 145% and 226%, respectively. In addition, organic acids generated by the degradation of lignin exerted influence on these enzymes, activated them.
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Palavras-chave
Celulases, Xilanases, Ligninases, Fermentação em estado sólido, Sacarificação enzimática, Etanol de segunda geração, Cellulases, Xylanases, Ligninases, Solid state fermentation, Enzymatic saccharification, Second generation ethanol